The adenosine diphosphate (ADP) receptor P2RY12 (purinergic receptor P2Y, G protein coupled, 12) plays a critical role in platelet aggregation, and P2RY12 inhibitors are used clinically to prevent cardiac and cerebral thrombotic events. Extracellular ADP has also been shown to increase osteoclast (OC) activity, but the role of P2RY12 in OC biology is unknown. Here, we examined the role of mouse P2RY12 in OC function. Mice lacking P2ry12 had decreased OC activity and were partially protected from age-associated bone loss. P2ry12 -/-OCs exhibited intact differentiation markers, but diminished resorptive function. Extracellular ADP enhanced OC adhesion and resorptive activity of WT, but not P2ry12 -/-, OCs. In platelets, ADP stimulation of P2RY12 resulted in GTPase Ras-related protein (RAP1) activation and subsequent α IIb β 3 integrin activation. Likewise, we found that ADP stimulation induced RAP1 activation in WT and integrin β 3 gene knockout (Itgb3 -/-) OCs, but its effects were substantially blunted in P2ry12 -/-OCs. In vivo, P2ry12 -/-mice were partially protected from pathologic bone loss associated with serum transfer arthritis, tumor growth in bone, and ovariectomy-induced osteoporosis: all conditions associated with increased extracellular ADP. Finally, mice treated with the clinical inhibitor of P2RY12, clopidogrel, were protected from pathologic osteolysis. These results demonstrate that P2RY12 is the primary ADP receptor in OCs and suggest that P2RY12 inhibition is a potential therapeutic target for pathologic bone loss. IntroductionOsteoclasts (OCs) are multinucleated myeloid lineage cells that are the principal source of bone resorptive activity (1). Enhanced OC activity, bone loss, and fractures are associated with rheumatoid arthritis, postmenopausal osteoporosis, and bone metastases (2). Modulation of osteoclastic bone resorption represents an attractive point of therapeutic intervention for the treatment of such conditions.Numerous purinergic G-protein-coupled nucleotide receptors are expressed in the bone microenvironment (3, 4). For example, uridine diphosphate-activated (UDP-activated) P2Y6 has been reported to increase NF-κB activation and OC survival (5), while P2Y2 (an ATP receptor) expression on osteoblasts (OBs) blocks bone mineralization (6, 7). Hoebertz et al. demonstrated that extracellular adenosine diphosphate (ADP) stimulates OC bone resorption in vitro, in part through the ADP receptor P2Y1 on OC (8); however, other ADP receptors, including purinergic receptor P2Y, G protein coupled, 12 (P2RY12), which is the target of the widely prescribed antiplatelet drug clopidogrel (Plavix), have not been evaluated for their roles in osteoclastic bone resorption.

One in 20 carriers of human T-cell leukemia virus type 1 (HTLV-1) will develop adult T-cell leukemia/lymphoma (ATL), a disease frequently associated with hypercalcemia, bone destruction, and a fatal course refractory to current therapies. Overexpression of the HTLV-1-encoded Tax oncoprotein under the human granzyme B promoter causes large granular lymphocytic leukemia/lymphomas in mice. We found that Tax ؉ mice spontaneously developed hypercalcemia, highfrequency osteolytic bone metastases, and enhanced osteoclast activity. We evaluated Tax tumors for the production of osteoclast-activating factors. Purification of Tax ؉ tumor cells and nonmalignant tumor-infiltrating lymphocytes demonstrated that each of these populations expressed transcripts for distinct osteoclastactivating factors. We then evaluated the effect of osteoclast inhibition on tumor formation. Mice doubly transgenic for Tax and the osteoclast inhibitory factor, osteoprotegerin, were protected from osteolytic bone disease and developed fewer soft-tissue tumors. Likewise, osteoclast inhibition with bone-targeted zoledronic acid protected Tax IntroductionAdult T-cell leukemia/lymphoma (ATL) is an aggressive lymphoproliferative malignancy of helper T lymphocytes and is associated with hypercalcemia and osteolytic bone lesions. 1 Human T-cell leukemia virus type 1 (HTLV-1), the first pathogenic retrovirus discovered in humans, is the etiologic agent of tropical spastic paraparesis myelopathy and ATL. 2,3 It is estimated that 10 to 20 million people worldwide are infected with HTLV-1. Infection with HTLV-1 in infancy is a major risk factor for the development of leukemia. 4 Approximately 1 in 20 people infected with HTLV-1 develop ATL at 20 to 60 years of age, indicating a long latency for this disease. The viral regulatory protein, Tax, has been implicated in the pathogenesis of ATL. [5][6][7] Tax is a transcriptional activator that can transactivate several viral and cellular genes encoding proteins such as interleukins 6 and 1 (IL-6 and IL-1), tumor necrosis factor ␣ (TNF␣), transforming growth factor-␤ (TGF␤), and parathyroid hormone-related peptide (PTHrP). 8,9 Tax is an oncoprotein with a capacity to transform cells, inhibit p53, and induce tumor formation in several transgenic mouse models using different tissuespecific promoters driving Tax expression. [10][11][12][13][14][15] Tax expression in mice under the HTLV-1 long terminal repeat or the metallothionein promoter did not produce leukemia; however, increased osteoclast resorption was noted. [15][16][17] Only the Tax transgenic mice under the regulation of the human granzyme B promoter (expressed in activated T and natural killer [NK] cells) develop lymphoproliferative disease (leukemia, lymphomas, splenomegaly) similar to ATL.Tax ϩ mice predominantly develop the large granular leukemia/ lymphoma (LGL) phenotype rather than the CD4 ϩ lymphoma/ leukemia most commonly seen in ATL. 18 Furthermore, Tax ϩ mice have been used to define cooperating genes critical to lymphoproliferative disease dev...

osteoclasts, also rescued the osteoclast defect in CD47 À/À cells.We then examined the consequences of this osteoclast defect in bone metastasis. In a model of tumor metastasis to bone, bone tumor burden was decreased in the CD47 À/À mice compared with wild-type (WT) controls, with no decrease in s.c. tumor growth in CD47 À/À mice. There was decreased tumor-associated bone destruction in the CD47 À/À mice compared with WT controls, consistent with a defect in osteoclast function that was not rescued by the presence of tumor. Our data show that CD47 regulates osteoclastogenesis, in part, via regulation of NO production, and its disruption leads to a decrease in tumor bone metastasis. CD47 is a novel therapeutic target to strengthen bone mass and diminish metastatic tumor growth in bone.

The purpose of this work was to determine platelet and myeloid cell-specific requirements for beta3-containing integrins in hemostasis, bone resorption, and tumor growth. LoxP-flanked mice were generated to study the conditional deletion of beta3-integrin in platelets [knockout in platelets (KOP)] and myeloid cells [knockout in myeloid (KOM)]. Using the beta3KOP and beta3KOM strains of mice, we studied the role of beta3-integrin in hemostasis, bone resorption, and subcutaneous tumor growth. Tissue-specific deletion of platelet beta3-integrins in beta3KOP mice did not affect bone mass but resulted in a severe bleeding phenotype. No growth difference of tumor xenografts or in neoangiogenesis were found in beta3KOP mice, in contrast to the defects observed in germline beta3(-/-) mice. Conditional deletion of myeloid beta3-integrins in beta3KOM mice resulted in osteopetrosis but had no effect on hemostasis or mortality. Tumor growth in beta3KOM mice was increased and accompanied by decreased macrophage infiltration, without increase in blood vessel number. Platelet beta3-integrin deficiency was sufficient to disrupt hemostasis but had no effect on bone mass or tumor growth. Myeloid-specific beta3-integrin deletion was sufficient to perturb bone mass and enhance tumor growth due to reduced macrophage infiltration in the tumors. These results suggest that beta3-integrins have cell-specific roles in complex biological processes.-Morgan, E. A., Schneider, J. G., Baroni, T. E., Uluçkan, O., Heller, E., Hurchla, M. A., Deng, H., Floyd, D., Berdy, A., Prior, J. L., Piwnica-Worms, D., Teitelbaum, S. L., Ross, F. P., Weilbaecher, K. N. Dissection of platelet and myeloid cell defects by conditional targeting of the beta3-integrin subunit.

Hedgehog (Hh) signaling is implicated in bone development and cellular transformation. Here we demonstrate that inhibition of Hh pathway activity inhibits tumor growth through effects on the microenvironment. Pharmacological inhibition of the Hh effector Smoothened (Smo) increased trabecular bone in vivo and inhibited osteoclastogenesis in vitro. In addition, enhanced Hh signaling due to heterozygosity of the Hh inhibitory receptor Patched (Ptch1+/−) increased bone resorption, suggesting direct regulation of osteoclast activity by the Hh pathway. Ptch1+/− mice had increased bone metastatic and subcutaneous tumor growth, suggesting that increased Hh activation in host cells promoted tumor growth. Subcutaneous growth of Hh-resistant tumor cells was inhibited by LDE225, a novel orally bioavailable Smo antagonist, consistent with effects on tumor microenvironment. Knockdown of the Hh ligand Sonic Hh (SHH) in these cells decreased subcutaneous tumor growth and decreased stromal cell production of IL-6, indicating that tumor-derived Hh ligands stimulated tumor growth in a paracrine fashion. Together our findings demonstrate that inhibition of the Hh pathway can reduce tumor burden, regardless of tumor Hh responsiveness, through effects on tumor cells, osteoclasts and stromal cells within the tumor microenvironment. Hh may be a promising therapeutic target for solid cancers and bone metastases.

This article describes the evaluation of the radiopharmaceutical 64 Cu-CB-TE2A-c(RGDyK) ( 64 Cu-RGD) as an imaging agent for osteolytic bone metastases and their associated inflammation by targeting of the a v b 3 integrin on osteoclasts and the proinflammatory cells involved at the bone metastatic site. Methods: The 64 Cu-RGD radiotracer was evaluated in the transgenic mouse expressing Tax (Tax 1 ), which spontaneously develops osteolytic tumors throughout the vertebrae and hind limbs, using biodistribution studies and small-animal PET/CT. Histologic analysis was also performed on Tax 1 mouse tails, using hematoxylin and eosin and tartrate-resistant acid phosphatase to confirm the presence of osteolytic bone lesions and the presence of osteoclasts, respectively. Additionally, a proof-of-principle study was conducted with a small group of Tax 1 animals presenting with osteolytic lesions. These animals were treated with the bisphosphonate zoledronic acid and imaged with 64 Cu-RGD to determine whether this radiopharmaceutical was sensitive enough to detect a response to the bisphosphonate therapy. Results: Biodistribution studies using 64 Cu-RGD demonstrated that Tax 1 mice between the ages of 6 and 12 mo had a greater accumulation of activity in their tail vertebrae than did the wildtype (WT) cohort (P 5 0.013). Additionally, Tax 1 mice between the ages of 6 and 12 mo had significantly more tracer activity associated with their tail vertebrae than did Tax 1 mice older than 12 mo (P 5 0.003), suggesting that earlier bone metastases cause an increased recruitment of a v b 3 -expressing cells. Small-animal PET/CT with 64 Cu-RGD was conducted on Tax 1 and WT mice. On the basis of standardized uptake value analysis, Tax 1 mice had approximately 2-fold more tail-associated activity than did WT animals (P 5 0.0157). Additionally, decreases in uptake were observed in the tails of Tax 1 mice after treatment with the osteoclast inhibitor zoledronic acid, and histologic analysis of Tax 1 mouse-tail vertebrae revealed the presence of Tax 1 tumor cells, osteoclasts, and proinflammatory cells within the bone microenvironment. Conclusion: Together, these data suggest that 64 Cu-RGD has the potential to effectively image osteolytic bone metastases and monitor the physiologic changes in the bone metastatic microenvironment after osteoclast-inhibiting bisphosphonate therapy. Bone metastases are a debilitating form of metastases causing severe bone pain, hypercalcemia, and pathologic bone fracture, and in some cases paralysis. Although the number of Americans living with bone metastases is unknown, it is estimated that at least 25% of those who die of cancer have them at the time of death (1). Bone metastases are classified as either osteoblastic or osteolytic lesions. The former results from the stimulation and proliferation of osteoblasts and culminates in the formation of excess bone. Conversely, osteolytic lesions are caused by the vicious cycle of bidirectional interactions between tumor cells and osteoclasts. In this cycle, i...

Bisphosphonates (BPs), bone targeted drugs that disrupt osteoclast function, are routinely used to treat complications of bone metastasis. Studies in preclinical models of cancer have shown that BPs reduce skeletal tumor burden and increase survival. Similarly, we observed in the present study that administration of the Nitrogen-containing BP (N-BP), zoledronic acid (ZA) to osteolytic tumorbearing Tax + mice beginning at 6 months of age led to resolution of radiographic skeletal lesions. N-BPs inhibit farnesyl diphosphate (FPP) synthase, thereby inhibiting protein prenylation and causing cellular toxicity. We found that ZA decreased Tax+ tumor and B16 melanoma viability and caused the accumulation of unprenylated Rap1a proteins in vitro. However, it is presently unclear whether N-BPs exert anti-tumor effects in bone independent of inhibition of osteoclast (OC) function in vivo. Therefore, we evaluated the impact of treatment with ZA on B16 melanoma bone tumor burden in irradiated mice transplanted with splenic cells from src -/-mice, which have non-functioning OCs. OC-defective mice treated with ZA demonstrated a significant 88% decrease in tumor growth in bone compared to vehicle-treated OC-defective mice. These data support an osteoclastindependent role for N-BP therapy in bone metastasis.

Interferon-␥ (IFN-␥ IFN-␥3 is a multifunctional cytokine produced mainly by NK cells and activated T cells that plays a critical role in host immune responses against pathogens and cancer (1). Mice deficient in IFN-␥, the R1 subunit of the IFN-␥ receptor, or the transcription factor STAT1 are more susceptible to spontaneous tumor development (1-3). IFN-␥ has also been found to have direct anti-proliferative and pro-apoptotic effects on tumor cells in animal models (4, 5); however, administration of high dose IFN-␥ to patients with advanced renal and ovarian cancer has had only limited success and failed to improve overall survival (1, 6).IFN-␥ has been shown to regulate bone cell differentiation and function with complex effects on skeletal health. However, the role of IFN-␥ in pathological bone disease is largely controversial. Previously, it has been reported that IFN-␥ can inhibit the critical osteoclast regulator, receptor activator of NFB ligand (RANKL), by activating ubiquitin-mediated degradation of its signaling pathway adaptor protein 8). Mice deficient for IFN-␥ or its receptor develop enhanced bone loss associated with collagen-induced arthritis (9 -11). In contrast, Gao et al. (12) recently found that IFN-␥ indirectly stimulates osteoclast formation and bone loss after ovariectomy via antigen-driven T cell activation, resulting in the production of osteoclast-activating factors. Interestingly, IFN-␥ has been used to treat infantile osteopetrosis in which patients suffered from high bone mass secondary to osteoclast dysfunction or osteoblast hyperactivity, but the mechanism of action may be through modulation of the host immune system rather than direct effects on bone cells (13-15). However, the role of IFN-␥ in the treatment of osteolytic bone metastases has not been elucidated.We evaluated the effects of IFN-␥ in HTLV-1-Tax transgenic mice that develop osteolytic bone tumors and hypercalcemia (16,17). Previously, it was shown that HTLV-1-Taxmice develop increased numbers of soft tissue tumors with enhanced tumor-associated angiogenesis and up-regulation of vascular endothelial growth factor expression; however, the impact on bone metastases and hypercalcemia in these mice * This work was supported, in whole or in part, by National Institutes of Health Grants PPG CA100730 and R01 CA 097250 (to Z. X., M. H., H. D., M. C. E., D. H. F., E. A. H., and K. N. W.), Grant T32 CA09547 (cancer biology training grant to O. U. and M. H.), and Grant RO1 CA100730 (to T. J. R. and W. P. D. and NCRR to S. S.), as well as by the St. Louis Men's Club Against Cancer (to K. N. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.